SARS-CoV-2 molecular epidemiology in Slovenia, January to September 2021

Background Sequencing of SARS-CoV-2 PCR-positive samples was introduced in Slovenia in January 2021. Our surveillance programme comprised three complementary schemes: (A) non-targeted sequencing of at least 10% of samples, (B) sequencing of samples positive after PCR screening for variants of concern (VOC) and (C) sequencing as per epidemiological indication. Aim We present the analysis of cumulative data of the non-targeted surveillance of SARS-CoV-2 and variant-dependent growth kinetics for the five most common variants in Slovenia for the first 9 months of 2021. Methods SARS-CoV-2 PCR-positive samples, from January to September 2021, were selected for sequencing according to the national surveillance plan. Growth kinetics studies were done on Vero E6 cells. Results Altogether 15,175 genomes were sequenced and 64 variants were detected, of which three successively prevailed. Variant B.1.258.17 was detected in ca 80% of samples in January and was replaced, within 9 weeks, by the Alpha variant. The number of cases decreased substantially during the summer of 2021. However, the introduction of the Delta variant caused a fourth wave and completely outcompeted other variants. Other VOC were only detected in small numbers. Infection of Vero E6 cells showed higher replication rates for the variants Alpha and Delta, compared with B.1.258.17, B.1.258, and B.1.1.70, which dominated in Slovenia before the introduction of the Alpha and Delta variants. Conclusion Information on SARS-CoV-2 variant diversity provided context to the epidemiological data of PCR-positive cases, contributed to control of the initial spread of known VOC and influenced epidemiological measures.

implementing the following criteria: Lineage -B.1.258.17, Completeyes, High coverageyes. Based on amino-acid substitutions we made a seleciton of 73 most prevalent sub-lineages (n total >= 10 entries) and calculated philogenetic clustering using 'ward.D' clustering method and 'correlation' distances (R package 'pvclust'). For each sub-lineage we present number of GISAID entries per country (heat-plot) and the information of the country and sample collection date (year-month) for the first uploaded instance of respected sub-lineage.

Library preparation, sequencing, and sequence analysis
Illumina compatible libraries were prepared with target genome amplification either with the ARTIC v3 protocol (URL: dx.doi.org/10.17504/protocols.io.bbmuik6w) or QIASEQ SARS-CoV-2 Primer Panel (QIAGEN). Both protocols were used according to published guidelines and recommendations or manufacturers manual. Partial library preparation followed by sequencing and bioinformatic analysis were performed at the Clinical Institute of Special Laboratory Diagnostics, University Children's Hospital, University Medical Center Ljubljana. Sequencing was performed on Illumina platforms, either MiSeq or NovaSeq 6000. The choice of kits and sequencing platforms depended on reagent availability. Sequencing was partially provided also by ECDC (n = 1,725 genomes).
Consensus genome sequences were obtained by 1) mapping reads to the reference genome (NC_045512) with BWA aligner (v 0.7.17) and 2) implementing the ivar (v 1.3)/samtools (mpileup, v 0.1.19) procedure for quality filtering, removing primer binding sites, and producing consensus sequences. SARS-CoV-2 variants were called with locally installed Pangolin (daily updated version; (3)), and mutations were obtained with online Nextclade analysis (4). Comparative genomics was performed by full genome alignment with mafft (v 7.480) and tree calculation with the IQ-TREE (v 1.6.1) GTR+G model. An exception was phylogenetic analysis of lineage B.1.258.17, for which a complete set of 6987 sequences belonging to the B.1.258.17 lineage was obtained from GISAID (20.5.2022) implementing the following criteria: Lineage -B.1.258.17, Complete -Yes, High coverage -Yes. Based on amino-acid substitutions, a selection of the 73 most prevalent sub-lineages was made (n total ≥ 10 entries), and phylogenetic clustering was performed using the 'ward.D' clustering method and 'correlation' distances (R package 'pvclust').

Virus isolation and growth kinetics using Vero E6 cells
Vero E6 cells were maintained in Eagle's Minimum Essential Medium (EMEM, ATCC 30-2003) containing 10% FBS (Gibco) and 1% antibiotic-antimycotic (100X, Gibco) at 37 °C and 5% CO2. Cells were routinely tested for mycoplasma and remained mycoplasma-free. All work with viruses in cell cultures was performed in a biosafety level 3 laboratory (BSL3) at the Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana.
Samples of variants B.1.258.17 (n=6), B.1.258 (n=4), B.1.1.70 (n=3), Alpha/B.1.1.7 (n=3), and Delta/AY.43 sublineage (n=5) were included in the growth kinetics experiment. SARS-CoV-2 variants were isolated on Vero E6 cells from samples collected from SARS-CoV-2-positive individuals. Two or three passages of SARS-CoV-2 isolates were performed to prepare the working virus stock. Cytopathic effects were observed under an inverted microscope (Eclipse Ts2R, Nikon). The virus titers of the working stocks were determined by titration of the virus isolates on Vero E6 cells and measured as 50% infectious tissue culture dose. To confirm the genomic sequences of the viral variants, RNA was extracted from the cell culture supernatant using the MagMAX™ CORE Nucleic Acid Purification Kit on the KingFisher Flex System (Thermo Scientific), and WGS was performed.
For the virus growth kinetics experiments, Vero E6 cells were seeded (3.5 × 10 5 cells/well) into 96-well microtiter plates and incubated at 37 °C and 5% CO2. After 1 day, the cell monolayers were inoculated with 500 of 50% infectious tissue culture dose TCID50 of SARS-CoV-2 variants. Supernatants were collected at 2, 24, 72, and 120 h after inoculation. RNA was extracted from the supernatants using the MagMAX™ CORE Nucleic Acid Purification Kit on the KingFisher Flex System (Thermo Scientific). Each viral variant was tested in six replicates for each time point, and viral load from the supernatants was measured using the SARS-CoV-2 real-time PCR assay targeting the E gene (5).
All Cq values were normalized against measurements obtained after 2 h of incubation (considered as a baseline viral load), according to the equations (1) and (2) shown below.
(1) Weight (sample) = Cq (sample at 2 h) / Cq min (2 h); where Cq (sample at 2 h) is the sample measurement at 2 h, and Cq min (2 h) is the lowest Cq measurement among all samples at the 2 h time-point.
(2) Normalized Cq (sample) = Cq (sample) / Weight (sample); where Cq (sample) is the Cq measurement of the sample at consecutive time points.